Adenovirus was known to have several advantages of mediating gene transfer with high efficiency in vivo and in vitro, transferring exogenous genes into a variety of cell types and allow for expression of the genes, regardless of cell division state of target cells, producing high-titer virus and not causing cancer in humans. Due to these advantages, adenovirus has greatly increased in use in clinical cancer gene therapy (Graham, F. L. ‘Adenovirus vectors for high-efficiency gene transfer into mammalian cells.’ Immunol. Today, 2000, 21, 426-8; Castell, J. V. et al. ‘Adenovirus-mediated gene transfer into human hepatocytes: analysis of the biochemical functionality of transduce cells.’ Gene Ther., 1997, 4, 455-64). When cancer is treated using gene therapy mediated by adenovirus, long term expression of therapeutic genes is not required, and host immune response induced by virus or viral proteins is not highly significant or even can be beneficial in some cases. Thus, adenovirus becomes attractive as a gene transfer vehicle for cancer therapy.
However, the most conventional recombinant adenoviruses for cancer therapy, which are known as replication-incompetent first-generation viruses, display antitumor activity in only primary infected cells or a very small number of surrounding cells (Vile, R. G. et al. ‘Cancer gene therapy: hard lesion and new courses.’ Gene Ther., 2000, 7, 2-8; Paillard, F. Cancer gene therapy annual conference. 1997: trends and news, Hum. Gene Ther., 1998, 4, 283-6; Lattime, E. C. et al. ‘Selectively replicating viruses as therapeutic agents against cancer.’ in: D. Kirn (Ed), Gene therapy of cancer, Academic Press, New York, 1999, 235-50).
To overcome such problems, the McCormick research group reported first a recombinant adenovirus that replicates selectively in tumor cells and eventually kills the tumor cells. After that, a variety of efforts were made to develop modified adenoviruses causing tumor-specific cytolysis (Bischoff, J. R. et al. ‘An adenovirus mutant that replicates selectively in P53-deficient human tumor cells.’ Science, 1996, 18, 274(5286), 373-6; Heise, C. et al. ‘ONYX-015, an E1B gene-attenuated adenovirus, causes tumor-specific cytolysis and antitumoral efficacy that can be augmented by standard chemotherapeutic agents.’ Nat. Med., 1997, 3(6), 639-45). The oncolytic adenoviral vectors have a domino effect on cancer therapy by displaying antitumor activity not only in primary infected cells, but also in secondarily infecting surrounding tumor cells by replication thereby remarkably increasing therapeutic efficacy against cancer. Also, the oncolytic adenoviral vectors further include the advantage of being inhibited replication in surrounding normal cells, and thus, having low cytotoxicity to the normal cells.
Tumor-specific replication-competent adenoviruses have been developed mainly by two methods, as follows. First, tumor-specific replication-competent adenoviruses can be developed by regulating cancer tissues expression of E1A protein which is essential for replication of adenovirus using tumor- or tissue-specific promoters. Rodriguez, R. et al. reported in 1997 that, when the upstream promoter of E1A gene is replaced by the promoter/enhancer region of prostate-specific antigen (PSA) selectively expressed in prostate cancer cells, adenovirus replicates selectively in PSA-positive prostate cancer cells (Rodriguez, R. et al. ‘Prostate attenuated replication competent adenovirus (ARCA) CN706: a selective cytotoxic for prostate-specific antigen-positive prostate cancer cells.’ Cancer Res., 1997, 1, 57(13), 2559-63). The prostate attenuated replication competent adenovirus (ARCA) CN706 (Calydon Pharmaceuticals, Calif., USA) is under a phase I clinical trial in patients suffering from recurred prostate cancer. In addition, some attempts have been made to develop more potent prostate cancer-specific replicative adenoviruses. For example, the expression of the two early genes, E1A and E1B, of adenovirus can be regulated according to expression levels of PSA by additionally inserting a prostate-specific enhancer region into the upstream of E1B gene that is one of early genes of adenovirus (Yu, D. C. et al. ‘Identification of the transcriptional regulatory sequences of human kallikrein 2 and their use in the construction of cyldon virus 764, an attenuated replication competent adenovirus for prostate cancer therapy.’ Cancer Res., 1999, 1, 59(7), 1498-504). Further, employing promoters of genes activated only in specific tumor cells, such as alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA) and MUC-1, tumor-specific replication-competent adenoviruses have been developed (Kanai, F. et al. ‘Gene therapy for alpha-fetoprotein-producing human hepatoma cells by adenovirus-mediated transfer of the herpes simplex virus thymidine kinase gene.’ Hepatology, 1996, 53, 963-7; Marshall, J. F. et al. ‘Tissue specific promoters in targeting systemically delivered gene therapy.’ Semin. Oncol., 1996, 23, 154-8; Osaki, T. et al. ‘Gene therapy for carcinoembryonic antigen-producing human lung cancer cells by cell type specific expression of herpes simplex virus thymidine kinase gene.’ Cancer Res., 1994, 54, 5258-61; Kurihara, T. et al. ‘Selectivity of a replication-competent adenovirus for human breast carcinoma cells expressing the MUC1 antigen.’ J. Clin. Invest., 2000, 106, 763-71).
In the second strategy for the development of tumor-specific replication-competent adenoviruses, some attempts have been tried to develop tumor-specific replication-competent adenoviruses by selectively knocking out adenoviral genes that are essential for active viral replication in normal cells but not essential in tumor cells (Whyte, P. et al. ‘Cellular targets for transformation by the adenovirus E1A proteins.’ Cell, 1989, 56, 67-75; Fueyo, J. et al. ‘A mutant oncolytic adenovirus targeting the Rb pathway produces anti-glioma effect in vivo.’ Oncogen., 2000, 19, 2-12). Bischoff J. R. et al. reported first in 1996 that an adenovirus mutant deficient in the adenoviral early protein E1B-55kD that functions to bind to and then inactivate the tumor suppressor protein p53 is capable of replicating selectively in p53 deficient tumor cells. When a wild-type adenovirus infects normal cells, the infected cells inhibit viral proliferation by activating the tumor suppressor protein p53. The E1B-55kD protein is responsible for the p53 activation, which binds to p53 and inhibits its function. As a result the wild-type adenovirus actively proliferates in the normal cells and eventually destroys the cells (Yew, P. R. et al. ‘Adenovirus E1B oncoprotein tethers a transcriptional repression domain to p53.’ Genes, 1994, 8, 190-202; Dobner, T. et al. ‘Blockage by adenovirus E4 or F6 of transcriptional activation by the p53 tumor suppressor.’ Science, 1996, 7, 272(5267), 1470-3.). However, when the E1B-55kD gene-deleted recombinant adenovirus infects normal cells, viral proliferation is inhibited because p53 inactivation is not induced, whereas the virus actively proliferates in several tumor cells in which the function of p53 is inhibited and eventually induces cell death of the infected cells. Based on these fact, the present inventors developed an E1B-55kD gene-deleted, tumor-specific cytolytic adenovirus, YKL-1, which was demonstrated to be superior to the conventional first-generation adenoviruses that is replication-deficient in transfection efficacy. YKL-1 also has the oncolytic effect against several human tumor cells (Lee, H. et al. ‘Oncolytic potential of E1B55kDa-deleted YKL-1 recombinant adenovirus: Correlation with p53 functional status.’ Int. J. Cancer, 2000, 88, 454-63).
In addition to inactivating p53, the E1B55kD protein stimulates to transport the adenovirus mRNA to the cytosol and synthesize the composed proteins of adenoviruses, and thus is essential for replication of adenoviruses. Therefore, the E1B55kD gene-deleted replication-competent adenovirus is proliferation-restricted in tumor cells and thus has reduced cytotoxic activity, resulting in a decrease in vivo antitumor efficacy. To solve this problem, the present inventors construct an Ad-ΔE1B19 adenovirus deleted for the adenovirus E1B19kD gene of which translational product functions to inhibit apoptosis, and revealed that the Ad-ΔE1B19 adenovirus has greatly enhanced cytolytic effect and in vivo antitumor effect (Kim, J. et al. ‘Evaluation of E1B gene attenuated replicating adenoviruses for cancer gene therapy.’ Cancer Gene Therapy, 2002, 9, 725-736). However, in this case that the E1B55kD gene is not deleted to achieve effective replication of adenovirus. Cell death is increased by elevated adenovirus replication, but the tumor-specific replication activity is lost. Thus, the Ad-ΔE1B19 adenovirus is required to have tumor specificity for use in cancer treatment by gene therapy.
Human telomere reverse transcriptase (hTERT) is one subunit of the telomerase holoenzyme that is involved in uniformly maintaining telomere length during chromosome replication, and known to be related to cell aging, tumorogenesis and cell immortalization (Counter, C. M. et al. ‘Telomere shortening associated with chromosome instability is arrested in immortal cell which express telomerase activity.’ EMBO J. 1992, 11, 1921-29; Kim, N. W. et al. ‘Specific association of human telomerase activity with immortal cells and cancer.’ Science, 1994, 21, 66, 2011-5; Harley, C. B. et al. ‘Telomeres shorten during aging of human fibroblasts.’ Nature, 1990, 345, 458-60). Telomerase activity is detected in germline cells and lymphocytes in human ovaries and testes, but not found in normal somatic cells (Wright, W. E. et al. ‘Telomerase activity in human germline and embryonic tissues and cells.’ Dev. Genet. 1996, 18, 173-9). Therefore, normal somatic cells have below a threshold length of telomere after a limited number of cell divisions, and eventually senesce (Yasumoto, S. et al. ‘Telomerase activity in normal human epithelial cells.’ Oncogene. 1996, 13, 433-9). In contrast, telomerase activity is elevated in benign tumor cells before tumor progression and cancer cells (Broccoli, D. et al. ‘Telomerase activity in normal and malignant hematopoietic cells.’ Proc. Nat'l. Acad. Sci. USA. 1995, 92, 9082-6.). After the first report in that telomerase activity is increased in ovarian cancer, elevated telomerase activity is detected in almost all human cancers, including blood cancer, stomach cancer, lung cancer, liver cancer, large intestine cancer, brain cancer, prostate cancer, head and neck cancer and breast cancer (Counter, C. M. et al. ‘Telomerase activity in human ovarian carcinoma.’ Proc. Nat'l. Acad. Sci. USA. 1994, 91, 2900-4; Counter, C. M. et al. ‘Stabilization of short telomeres and telomerase activity accompany immortalization of Epstein-Barr virus-transformed human B lympho-cytes.’ J. Virol., 1994, 68, 3410-4; Shay, J. W. et al. ‘A survey of telomerase activity in human cancer.’ Eur. J. Cancer, 1997, 33, 787-91; Harle-Bachor, C. et al. ‘Telomerase activity in the regenerative basal layer of the epidermis in human skin and in immortal and carcinoma-derived skin keratinocytes.’ Proc. Nat'l. Acad. Sci. USA. 1996, 93, 6476-81).
Expression of hTERT that plays a critical role in the function of telomerase is associated with telomerase activity. Recent data suggest that telomerase expression is regulated according to the activity of hTERT promoter, that is, mRNA levels of hTERT. The minimum hTERT promoter region to regulate hTERT activity is 181 bp in length. The wild-type hTERT promoter contains two c-Myc binding sites and five Sp1 binding sites. According to some reports the c-Myc oncoprotein which is highly expressed in tumor cells compared to normal cells is binding to the transcription factor Sp1. This binding activates hTERT promoter. Takakura M. et al. reported in 1999 that the activity of hTERT promoter is elevated by overexpression of c-Myc (Cerni, C. ‘Telomeres, telomerase, and myc.’ An update. Mutat. Res., 2000, 462, 31-47; Greenberg, R. A. et al. ‘elomerase reverse transcriptase gene is a direct target of c-Myc but is not functionally equivalent in cellular transformation.’ Oncogene, 1999, 18, 1219-26; Takakura, M. et al., ‘Cloning of human telomerase reverse transcriptase gene promoter and identification of proximal core promoter essential for transcriptional activation of hTERT in immortalized and cancer cells.’ Cancer Res., 1999, 59, 551-9).
As described by Shoji K. et al., apoptosis-inducing toxic genes such as caspase-8 can be expressed in only tumor cells by inducing expression of cancer cell-specific genes using the hTERT promoter (Shoji, K. et al. ‘A novel telomerase-specific gene therapy: gene transfer of caspase-8 utilizing the human telomerase catalytic subunit gene promoter.’ Human Gene Therapy, 2000, 11, 1397-406.). However, the use of the hTERT promoter alone has a limitation in attaining sufficient tumor specificity.